Minutes of the meeting held in Granada, September 25^th 1999

The annual meeting of the project FAIR CT 97 3039 was held at the Corrala de Santiago, Granada, Spain on Sept 25^th, 1999, hosted by Partner 2, M^a Dolores Suarez.

The meeting started at 3 pm with an introduction by the Coordinator Claudio Galli (Partner 1) who, on behalf of all the participants, thanked Maria Dolores Suarez (Partner 2) for the organization of the gathering.

Status of the project

The project has reached the stage of a) the the definition of the most appropriate procedure for the production of OMWW extracts to be applied to a pilot industrial plant and b) the assessment of the biological activities in vitro and in animals, since next year the project will be devoted to human studies. At this stage, it is necessary to set up a pilot plant for the production of phenolic extracts in the range of 200-300 g (containing at least 100 g of hydroxytyrosol).

Claudio Galli calculated in fact that 30-50 gr of extract would be necessary to carry out such studies. Such extract should be well-characterized in terms of the total phenol content/g of extract, the percentage of phenols, ortho-diphenols, and other constituents. Also, in order to submit an application to the ethic’s committee that will evaluate the feasibility of the incoming human studies, information on the phenolic content of olives, which are part of the Mediterranean diet, is needed. This is necessary in order to evaluate the correspondence between the amount of phenols to be administered to humans in the form of OMWW extracts – in terms of the amount of olives providing the same quantity of phenols.

Leonardo Setti (Partner 5), illustrated the different extraction procedures that they employed to optimize the recovery of phenolics from olive mill waste waters (OMWW). In particular, Partner 5 employed solid-liquid extraction, liquid-liquid extraction, and adsorption techniques with the use of resins. The first one is the most effective but is also very expensive, so participants agreed that adsorption of phenolic with ad hoc resins should be the technology of choice.

Claudio Galli showed the chromatographic analyses of the extracts that have been employed to carry out the second of the four parts of the project, i.e. in vivo studies in animal models. These were characterized by a high content in HT.

Nadia Mulinacci (Partner 4), illustrated the different technologies they employed to recover the highest amount of phenolics and concluded that the adsorption techniques is the most efficient one.

A general discussion took place concerning the necessity of knowing the proportion of phenols in the extract with respect to the total weight of the extract. Such data are not available at the moment but can be easily obtained. Also, participants discussed on the most appropriate vehicle to administer the extract to human subjects. Given the solubility of the extract, a mixture of water:ethanol 9:1 (vol:vol) will likely be employed. Finally, the proportion of phenols to be employed in encapsulated form should also be clearly defined and will likely be standardized at 10% of HT over the total capsule content (1 gr).

Leonardo Setti (Partner 5), stated that the adsorption technique, although well-established, could be further improved to increase the yield and said that he’s group will work on this issue in the immediate future.

Joao Bordado (Partner 7) reported on the costs of the recovery of active compounds from OMWW. As it would be too expensive to transport OMWW from the mill to the extraction plant, the best solution is likely to pump OMWW into a drum containing the resins, that can be subsequentely shipped to the manufacture plant. Important issues to be taken into account include the overall yield, the possibility to reuse and reactivate the resins. From 1 lt of OMWW, he estimated a recovery of ~ 1 gr of raw material containing ~ 130 mg of hydroxytyrosol.

Claudio Galli underlined that it is important to know if there is an analogy between the minor components of olives and those of OMWW, in order to strengthen the proposal to the ethic’s committee (see above).

Nadia Mulinacci replies that some compounds are indeed different but yet to be identified. Conversely, there is analogy between olive oil and OMWW minor constituents.

A general discussion took place concerning the use of enzymes to treat OMWW prior to the addition of resins. Such treatment would increase yield but also generate additional, unidentified compounds and increase costs. The group concluded that the use of enzyme is not worthy.

Claudio Galli extensively illustrated the results obtained by his group during the second year of the project, devoted to the study of OMWW in animal models. Such results will be included in the final report. These results represent a very solid bases for studies in humans, since the parameters tested are simple, mostly non-invasive (analyses of urinary metabolites and products) and very representative of the processes (antioxidant activities) to be investigated.

The discussion was interrupted at 4:55 pm for a coffee break and resumed at 5:20 pm.

Raffaello Prugger talked about the intellectual property of the project results, asking that results be made available to all participants prior to publication. Everyone agreed on this point.

Claudio Galli asked F.F. Vincieri to analyze the raw material that will be prepared by Partner 6 before proceeding with the human study. It was also recommended that comparative analyses of small aliquots of different OMWW samples be carried out before selecting the large (~ 500 lt) batch for the preparation.

Leonardo Setti suggested that, in order to optimize the extraction procedure, he should be able to employ OMWW different from those previously collected by Partner 6.

Claudio Galli asked that the two OMWW be very similar in composition.

Claudio Galli and Claude Leger discussed about the amount of phenolic compounds to be given to human subjects and concluded that the optimal dose should be 300 mg/day, of which 100 mg should be HT.

Claudio Galli asked F.F. Vincieri if it were possible to measure the total amount to hydroxytyrosol in OMWW, including both the free and the conjugated ones. F.F. Vincieri and N. Mulinacci answered that they will check into this possibility but think it possible.

Claudio Galli stated that 1 mg/Kg body weight of pure hydroxytyrosol should be the dose of choice, as it approximates the human daily intake, although the applicaytion to the ethic’s committee should read "100 mg/subject/day".

Maria Dolores Suarez (Partner 2) asked PG Pifferi to prepare some more fractions replicating those provided to the Coordinator’s lab and labeled A and B. L Setti agreed.

There was general agreement on the dose of 100 mg hydroxytyrosol/subject/day. The total weight of the extract to be administered daily as a pill should not exceed 1 gr.

Claude Leger (Partner 3) raised the issue of the appropriate placebo. This will be privately discussed with the Coordinator.

Olga Martinez Augustin (Partner 2) illustrated the results she obtained during the past year by studying the effects of OMWW phenols on liver enzymes involved in antioxidant defenses in the rat. A full report will be included in the final yearly report.

Claudio Galli illustrated the human studies his group will perform in the next year, according to the project’s approved protocol.

Francesco Visioli (Partner 1) summarized the main points that were discussed during the meeting and reviewed the agreements.

The meeting ended at 6:20 pm

• Claudio Galli, Francesco Visioli (Partner 1)
• Raffaello Pruger (Partner 2)
• Olga Martinez, Jose’ Juan Caballero, Victor Puerta, M^a Dolores Suarez, M^a Dolores Giron (Partner 2)
• Pier Giorgio Pifferi, Elena Arcosi, Leonardo Setti (Partner 5)
• Annalisa Romani, Nadia Mulinacci, Franco F. Vincieri (Partner 4)
• Catarina Lima, Joao Bordado (Partner 6)
• Pilar Garcia Parega, Armando L. Cambil (Partner 7)

Oct 31^st: Final reports should be sent to Partner 1

Dec 31^st: Preparation of the capsules for human studies